Estructura porcentual del pasivo de las IFM

Impedance Variations of Spheroids Prepared by the Same Initial Cell Number and Growth Duration

It is possible to generate spheroids of a defined size using a defined cell seeding density and growth duration (chapter 6.1). Five MCF-7 spheroids of a batch, which were prepared in the same way (same seeding density / age) were measured sequentially and the impedance variation between these spheroids was estimated. Three batches were measured this way obtaining the results below:

 Within the respective spheroid batches the IZIrel values have a relative error of 12 – 16 %.

 Comparison of the results of three batches show no significant differences in impedance.

Variability in spheroid impedance likely comes from minor differences in the spheroid structure. Even though, spheroids originate from the same initial cell density and are measured at the same age they differ slightly in size, shape and internal composition (ratio of cells to extracellular matrix; amount of dead cells). No two spheroids are completely alike and consequently, also their measured impedance is fluctuating to a certain degree.

Impedance Variations of Spheroids Prepared by Different Initial Cell Numbers and the Same Growth Duration

In this experiment an impedimetric and microscopic analysis of MCF-7 spheroids from three different seeding densities (1000 / 3000 / 6000 cells/well) and consequently, different sizes was performed using the new device.

 With increasing spheroid size the impedance is decreased.

 With increasing spheroid size Rext and Rint are decreasing and Asph is increasing.

 With increasing spheroid size the penetration into the narrowed channel is reduced.

The impedimetric results were counter intuitive since it was expected that spheroids with the highest seeding density tested would show the highest impedance as well as extra- / intracellular resistance values. This was for example reported in the work of Thielecke et al. (2001) who performed EIS measurements in a capillary system with T47D human breast carcinoma spheroids. Via subsequent data fitting using a nonlinear least square fitting technique they determined the Rext value of bigger spheroids with diameters of

600 – 700 µm to be 92 k whereas smaller spheroids with diameters of 400 – 500 µm had a Rext value of only 18 k. The path of current flow is longer through the bigger spheroid and it has to cross more cell layers and consequently, it provides also a higher resistance than the smaller spheroid.

But obviously, there are other parameters that influence the impedance of the spheroid-loaded flow channel in this thesis. The microscopic study of the translocation of differently sized spheroids (Fig. 25) indicates that the small spheroids are able to adapt their shape to the dimensions of the narrow channel more easily than larger spheroids. A huge part of the small spheroids was inside the narrowed channel leading to smaller extracellular spaces (and higher Rext) and an increased effective length of the spheroid that had to be crossed by the current. Bigger spheroids showed less tendency for deformation and consequently, the spheroid part inside the narrow channel was significantly smaller (with lower Rext) offering more extracellular space where the current could pass the spheroid.

Another aspect which would explain the lower Rext value of the bigger spheroids could be that they possibly contain more dead cells than the small spheroids due to the nutrition and waste gradients in big spheroids (chapter 1.1.3). Therefore, the ratio of the volume of viable cells to the volume of the whole spheroid is decreasing, which is generally linked to a decrease in Rext (Thielecke et al., 2001).

Nevertheless, the results of the microscopic study clearly indicate that the position of the spheroid in the aperture has an influence on the spheroid impedance. A spheroid that is completely inside the central aperture thus, should lead to a higher impedance than a spheroid that is partly inside the aperture. In all impedance experiments using the flow channel the spheroid sizes are

selected in the way that they are only partly inside the aperture to ensure reproducible experiment conditions. Only in experiments where substances lead to the disintegration of the spheroid and consequently to a reduced spheroid diameter it is possible that the spheroid enters the aperture completely. In this case it might be possible that the high impedance signal, due to the maximal deformation of the spheroid, is overlapping the actually low impedance of the disrupted spheroid.

Impedance Variations Upon Spheroid Repositioning

Repositioning is performed when the solution in the flow channel has to be exchanged during an impedimetric analysis of a spheroid (chapter 5.1.3, 5.3.3). For this purpose, the influence of the repositioning process on the spheroid impedance was investigated. Five spheroids were analyzed performing three repositionings after the initial introduction into the channel.

 The relative error of IZIrel for the five spheroids ranges between 5 – 26 %.

 The mean relative impedance values of three spheroids measured in FC-2 are not significantly different. The mean relative impedance values of two spheroids measured in FC-3 are also not significantly different.

It is possible to reposition a spheroid for solution exchange without creating too strong deviations in impedance. If extraordinary deviations in impedance occur after repositioning however, this might hint to an irregular spheroid shape. These could lead to differences in spheroid impedance due to a different effective spheroid size that has to be overcome by the current. A strong decrease in impedance after spheroid repositioning could indicate a damage of the spheroid during its removal before repositioning. To avoid such incidents the repositioning has to be performed very cautiously.

Another group also reported of a variance in impedance for identical spheroids. Krinke et al. (2010) performed a similar experiment using their impedance-based microcavity array (chapter 1.3). They measured the same spheroid 15 times and evaluated the corresponding average relative impedance (~ 50 %) observing a standard deviation of ~ 20 %. This would result in a relative error of ~ 40 %. In the study of this thesis typical relative impedance values were higher ranging

between ~ 300 % and ~ 1000 % and the corresponding standard deviations were ranging between ~ 20 % and ~ 200 %. The relative errors of IZIrel range between 5 – 26 % and are, thus, smaller than those reported of Krinke et al. (2010).

A general phenomenon in the experiments of this thesis (as well as for the repositioning experiment) is that the impedance values after spheroid introduction are clearly different for different PT-2 flow channels. For this purpose, the results from different channels were evaluated separately in this thesis. The reason for the distinct differences in impedance values could not be resolved. It is assumed that due to hair cracks in the channel bottom, which were seen in all four flow channels (Fig. 32, upper row), the bottom-surface around the aperture was roughened and thereby influenced the deformation of the spheroid into the aperture. Especially FC-2 shows a lot of cracks in comparison to FC-3 and might have resulted in the decreased impedance values. However, in the penetration of the spheroids into the central channel of FC-2, -3 and -4 no distinct differences could be observed (Fig. 32, lower row). The spheroid in FC-1 is the only one that shows a reduced penetration of the narrow channel.

Fig. 32: Confocal fluorescence micrographs of FITC-filled FC-1 – FC-4 showing hair cracks in the base material of the channel. These appear black in front of the green background from the FITC solution. Beneath the respective flow channel images corresponding phase contrast micrographs of 3000 cells/well spheroids are shown that are positioned at the aperture and partly inside the narrowed space.

5.5.3 Visualization of Spheroids in the Flow Channel

A spheroid positioned in the novel channel setup can be imaged with most conventional microscope types used for visualization of spheroids and cell monolayers. The use of transparent electrode and channel materials enables:

 Spheroid observation during an impedance measurement.

 Documentation of spheroid characteristics such as shape and position by phase contrast and epifluorescence micrographs.

 Visualization of internal structures of the spheroid by CLSM, which is however limited (microscopic limitations and channel related challenges).

 Spheroid exposure to excitation light for phototoxicity studies.

Overall, the novel EIS-based flow channel has a lot of advantages discussed in the previous chapters, enabling the sensitive impedimetric readout of 3D multicellular spheroids and the microscopic visualization at the same time.

6

Impedimetric Model Studies with Tumor

Spheroids

Morphological alterations of cells upon exposure to external stimuli can be detected sensitively using impedance spectroscopy. This was proven especially for 2D monolayer cells investigated by ECIS technology (Wegener et al., 2000; Stolwijk et al., 2011; Lieb et al., 2016). For 3D multicellular spheroids, however, appropriate EIS measurement devices are still in development (chapter 1.3). In this thesis a novel impedance-based device is introduced (chapter 5) and tested on spheroids.

Distinct morphological alterations are often observed in conjunction with cellular stress, injuries or cell death. In tumor therapy for example, these alterations are induced by potential therapeutic drugs, heat or irradiation. In this thesis the tumor spheroids formed from MCF-7 breast cancer cells (chapter 4.1.1) were used as 3D tissue models to study the morphological alterations upon different invasive external stimuli. First, their morphology and growth behavior is described (chapter 6.1). These characteristics are important information to generate spheroids with reproducible size being conform to the PT-2 flow channel geometries (chapter 5.1.1). In the following, proof-of-concept studies were performed using the new EIS-device investigating the response of MCF-7 spheroids to different stimuli with well-known effect. Upon exposure of spheroids to a chemical cross-linker (chapter 6.2), a detergent (chapter 6.3), buffers with different osmolarities (chapter 6.4) or an actin filament polymerization inhibitor (chapter 6.5) the spheroid morphology and structure is significantly altered. This way, dead spheroids with intact structure, spheroids with damaged membranes, swollen/shrinked spheroids and spheroids with disrupted actin filaments could be studied. Furthermore, the tumor spheroids were subjected to two cancer treatment therapies investigating the effect of hyperthermia (chapter 6.6) and photodynamic therapy (chapter 6.7).

The spheroidal response to the different stimuli was analyzed using impedance measurements as well as supplementary microscopic images, impedance measurements on cell monolayers or biochemical endpoint assays like the Presto

Blue® assay. Comparing the results of these additional methods the sensitivity and applicability of the newly designed device was evaluated.